Stabilization of the relative concentration of ATP and invariants in the regulation of erythrocyte energy metabolism

The problem is considered concerning the effect of individual differences of erythrocyte energetic metabolism parameters in the regulation (stablization) of ATP level. It has been found that equal quality of stabilization of relative ATP concentration (ATP/(AMP+ADP+ATP)) in erythrocytes with very different individual parameters is provided, firstly, by presence of scaling invariance of the characteristics of ATP-producing and ATP-consuming systems, and, secondly, by coincidence between the physiological values of ATP/(AMP+ADP+ATP) ratio for all the individuals. Scaling invariance property results in the essential reduction of the effective dimension of the individual parameters space. Revealed is a single parameter (function of all individual parameters), which defines the level of relative ATP concentration or the erythrocyte energy charge. The hypothesis is formulated that erythrocyte has two levels of energetic metabolism regulation, of which the first level is responsible for regulation of relative ATP concentration (due to the self-energy system functioning), while the second one provides (for a long time) regulation of the individual parameter values.     

Changes in the energy state of tissues in spontaneously hypertensive rats]

The contents of adenine nucleotides (ATP, ADP, AMP), phosphocreatine (PCr) and creatine (Cr) in the heart, skeletal muscle, liver and spleen in spontaneously hypertensive (SHR) and normotensive (WKY) rats. The ATP/ADP ratio in cardiac tissue was lower in SHR compared with WKY, while myocardial contents of adenine nucleotides, PCr and Cr did not differ significantly between the groups. A lower ATP/ADP ratio in the skeletal muscle SHR of was accompanied by a reduction of PCr content comparing with these indices in WKY rats. The liver and spleen of SHR exhibited lower ATP contents and higher ADP and AMP levels compared with those ones in WKY rats, despite of the close values of adenine nucleotide pools (sigma AN = ATP + ADP + AMP). This redistribution of tissue adenine nucleotides was corresponded to lower energy charges (EC = (ATP + 0.5 ADP)/sigma AN) and ATP/ADP ratios in SHR group. The reduction of the energy state of tissues in SHR rats increased in the following rank: heart > skeletal muscle > liver > spleen, thus, reflecting progressive decrease of intensity of oxidative metabolism. The results suggest changes in the balance of rates of ATP formation and hydrolysis occur at the system level in primary hypertension. Probably, consequences of such rearrangement in energy metabolism are functional disturbances of plasma membrane and sacroplasmic reticulum well-documented in a number of experimental and clinical studies.     

Luminometric assays of ATP, phosphocreatine, and creatine for estimation of free ADP and free AMP.

We present methods to measure ATP, phosphocreatine, and total creatine (the sum of creatine and phosphocreatine) in alkaline cell extracts. Knowledge of these parameters, together with the known equilibrium constants for the creatine kinase and adenylate kinase-catalyzed reactions, allows one to estimate the levels of free ADP and free AMP inside cells. The enzymatic assays for the above-mentioned metabolites all lead up to the production of ATP, which is measured luminometrically with the ATP-dependent oxidation of luciferin catalyzed by firefly luciferase. To determine phosphocreatine, endogenous ATP is first destroyed, and phosphocreatine is then quantitatively reacted with exogenous ADP to form ATP. Total creatine is measured after quantitative conversion of creatine to phosphocreatine with a large excess of exogenous ATP, conversion of all ATP to ADP, and final reaction of phosphocreatine with ADP to form ATP. We used 5-microl samples in 0.5-ml microcentrifuge tubes and subsequent 5-microl additions of analytical reagents. We expect that the volumes can be changed easily. We tested the methods with glucagon- and insulin-secreting cells. Estimates of free ADP and AMP are expected to be useful in many different areas of research, such as cellular energy metabolism, purine nucleotide metabolism, adenine nucleotide gating of ion channels, and release of vasoactive or angiogenic factors.     

ATP breakdown and resynthesis in the development of gastrointestinal mucosal damage and its prevention in animals and human (an overview of 25 years ulcer research studies).

The changes in membrane-bound ATP systems (breakdown and resynthesis) were analyzed in different experimental ulcer models (such as ETOH, HCl, NaOH, 25% NaCl-induced, pyloric ligated + epinephrine treated, stress, reserpine treated, indomethacin treated rat models) and chronic antral, duodenal and jejunal ulcers in patients. The energy system parameters (adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), cyclic AMP (cAMP), lactate) were measured from different sites of gastrointestinal mucosa, and values of ATP/ADP, adenylate pool (ATP + ADP + AMP) and energy charge ((ATP + 0.5 ADP)/(ATP + ADP + AMP)) were calculated. The biochemical measurements were done at different times during the development of gastrointestinal mucosal lesions, without and with application of different drugs (PGI2, atropine, cimetidine) and bilateral surgical vagotomy. The aims of our present paper were: 1.) To summarize the main directions of ATP breakdown during the development of gastrointestinal lesions or ulcers in different experimental models and human beings: 2.) To summarize the biochemical steps of defense of gastrointestinal mucosa against chemicals, drugs or unknown pathogenic factors; 3.) To analyze the importance of membrane-bound ATP-dependent energy systems in order to understand the mucosal lesions and their prevention; 4.) To evaluate the real values of changes in these parameters from the point of view of ulcerogenesis and its prevention; 5.) To find some correlation between the energy parameters during mucosal damage and its prevention: 6.) To understand better the types of tissue reactions (metabolic) due to development of mucosal lesions and prevention.(ABSTRACT TRUNCATED AT 400 WORDS)     

Energy aspects of the growth of Escherichia coli synchronized by starvation

The quantitative determination of adenyl nucleotides based on the separation of their dansyl derivatives by thin layer chromatography has made it possible to study the dynamics of changes in the pool of ATP, ADP and AMP in Escherichia coli K-12 during its synchronous growth after glucose starvation. The energy parameters (the adenylate pool, energy charge, teh ATP/ADP ratio, the rates of oxygen uptake and ATP generation, the economic coefficients of oxygen and ATP utilization) were compared with changes in the growth characteristics (the rate of growth and biomass concentration). This comparison allowed the authors to draw the conclusion about the uncoupled constructive and energy metabolism and about the possible regulatory role of energy parameters in the synchronised culture growth.     

Adenylic nucleotides and energy charge during the embryonic development of Bufo arenarum

The contents of ATP, ADP and AMP were determined by HPLC and adenylic energy charge (AEC) was estimated during different stages of the embryonic development of Bufo arenarum up to the tailbud stage. All the developmental stages studied showed a high ATP content (about 1.04-1.48 nmol/emb.). The concentration of ADP was low (0.025-0.041 nmol/emb.) but rose slightly at the neural tube stage. AMP was undetectable before the tailbud stage. AEC values were almost constant (about 0.987-0.992) throughout the period studied. Only a fall at the tailbud stage could be detected which can be related to this more advanced cellular differentiation stage.     

Cellular energy systems and reserpine ulcer in rats

Gastric ulcer was elicited in rats by reserpine (5 mg x kg-1 sc.) administration. Ulcer formation (number and severity) was measured 6, 12, 18 and 24 hr after reserpine administration. At the time of killing of the animals, tissue levels of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), cyclic adenosine monophosphate (cAMP) were measured enzymatically and by radioimmunoassay in the gastric fundal mucosa. The sum of ATP + ADP + AMP (adenylate pool) and the ratio of ATP x ADP-1 were calculated. It was found that (1) the tissue levels of ATP, AMP, cAMP, sum of ATP / ADP + AMP (adenylate pool) and ratio of ATP x ADP-1 increased significantly in the gastric fundal mucosa 6 hr after reserpine administration, thereafter these values decreased gradually and significantly; (2) the tissue level of ADP increased significantly in the gastric fundal mucosa 6 hr after reserpine administration, meanwhile its level increased significantly at 18 and 24 hr; (3) the value of energy charge (ATP + 0.5 ADP x ATP + ADP + AMP-1) remained unchanged; (4) the peaks of biochemical alterations in the gastric fundus mucosa preceded he appearance of ulcers. It was concluded that (1) reserpine ulcer appears after an active metabolic response in the rat gastric fundal mucosa; (2) hypoxaemic damage in the gastric fundal mucosa can be excluded as a possible underlying mechanism of ulcer formation produced by reserpine administration; (3) before the appearance of reserpine ulcer, significant changes in the feedback mechanism, system, i.e. between the ATP--membrane ATPase--ADP and the ATP--adenylate cyclase--cAMP energy systems, can be observed in the rat gastric fundal mucosa.     

Energy status in the fat body of Trichoplusia ni parasitized by the insect parasite Hyposoter exiguae

The levels of adenylate nucleotides were examined in 4th-instar Trichoplusia ni larvae 3 days after parasitization by the insect parasite Hyposoter exiguae. In general, parasitization caused a decrease in the level of ATP and increased ADP and AMP levels. These changes resulted in alteration of the adenylate kinase mass-action ratio. The overall energy status of parasitized larvae, however, as indicated by energy ratios, including the “energy charge,” was affected only slightly. The result demonstrates that the host maintained an active and viable metabolic state despite extensive alterations in physiology which occur at this stage of the parasite-host association.     

Functional Characterization of TbMCP5, a Conserved and Essential ADP/ATP Carrier Present in the Mitochondrion of the Human Pathogen Trypanosoma brucei

Trypanosoma brucei is a kinetoplastid parasite of medical and veterinary importance. Its digenetic life cycle alternates between the bloodstream form in the mammalian host and the procyclic form (PCF) in the bloodsucking insect vector, the tsetse fly. PCF trypanosomes rely in the glucose-depleted environment of the insect vector primarily on the mitochondrial oxidative phosphorylation of proline for their cellular ATP provision. We previously identified two T. brucei mitochondrial carrier family proteins, TbMCP5 and TbMCP15, with significant sequence similarity to functionally characterized ADP/ATP carriers from other eukaryotes. Comprehensive sequence analysis confirmed that TbMCP5 contains canonical ADP/ATP carrier sequence features, whereas they are not conserved in TbMCP15. Heterologous expression in the ANC-deficient yeast strain JL1Δ2Δ3u⁻ revealed that only TbMCP5 was able to restore its growth on the non-fermentable carbon source lactate. Transport studies in yeast mitochondria showed that TbMCP5 has biochemical properties and ADP/ATP exchange kinetics similar to those of Anc2p, the prototypical ADP/ATP carrier of S. cerevisiae. Immunofluorescence microscopy and Western blot analysis confirmed that TbMCP5 is exclusively mitochondrial and is differentially expressed with 4.5-fold more TbMCP5 in the procyclic form of the parasite. Silencing of TbMCP5 expression in PCF T. brucei revealed that this ADP/ATP carrier is essential for parasite growth, particularly when depending on proline for energy generation. Moreover, ADP/ATP exchange in isolated T. brucei mitochondria was eliminated upon TbMCP5 depletion. These results confirmed that TbMCP5 functions as the main ADP/ATP carrier in the trypanosome mitochondrion. The important role of TbMCP5 in the T. brucei energy metabolism is further discussed.     

Regulation of human neutrophil functions by adenine nucleotides

Previous work has shown that platelet-derived adenine nucleotides modulate neutrophil superoxide anion (O2-) generation. Additional studies were undertaken to characterize the effects of authentic adenosine (ADO) and its nucleotide derivatives on the inflammatory functions of human neutrophils. Stimulus-specific inhibition of neutrophil O2- generation by ADO in response to FMLP was verified. In addition, the ability of ATP, ADP, and AMP to limit neutrophil O2- generation induced by FMLP (0.2 to 0.5 microM) was demonstrated. The concentration producing 50% inhibition for nucleotide inhibition of neutrophil O2- generation was in the rank order of ADO (0.1 microM) less than AMP (0.5 microM) less than ADP less than or equal to ATP (5 microM). Guanine and inosine nucleotides (0.01 to 100 microM) did not inhibit FMLP-stimulated neutrophil O2- generation. Neutrophil degranulation in response to FMLP was only modestly inhibited by adenine nucleotides and ADO. Adenosine and ADP failed to affect chemotaxis of neutrophils stimulated with FMLP. The inability of non-metabolizable analogs to mimic the inhibitory effects of authentic ATP or ADP on the neutrophil O2- response suggested that metabolism of added nucleotides is necessary for their effectiveness. Both TLC and HPLC confirmed that ATP and ADP were converted to AMP and ADO after their incubation with unstimulated or FMLP-activated neutrophils. The addition of adenosine deaminase to neutrophil reaction mixtures in which conversion of added nucleotides was apparent removed detectable ADO but failed to completely abrogate the inhibition of neutrophil O2- generation by accumulated AMP. The kinetics of inhibition of FMLP-induced neutrophil O2- generation by ATP and ADP also indicated that conversion of these nucleotides to ADO and/or AMP may be essential for their ability to reduce neutrophil responses.     

Thermal Stress Promotes Host Mitochondrial Degradation in Symbiotic Cnidarians: Are the Batteries of the Reef Going to Run Out?

The symbiotic relationship between cnidarians and their dinoflagellate symbionts, Symbiodinium spp, which underpins the formation of tropical coral reefs, can be destabilized by rapid changes to environmental conditions. Although some studies have concluded that a breakdown in the symbiosis begins with increased reactive oxygen species (ROS) generation within the symbiont due to a decoupling of photosynthesis, others have reported the release of viable symbionts via a variety of host cell derived mechanisms. We explored an alternative model focused upon changes in host cnidarian mitochondrial integrity in response to thermal stress. Mitochondria are often likened to being batteries of the cell, providing energy in the form of ATP, and controlling cellular pathway activation and ROS generation. The overall morphology of host mitochondria was compared to that of associated symbionts under an experimental thermal stress using confocal and electron microscopy. The results demonstrate that hyperthermic stress induces the degradation of cnidarian host mitochondria that is independent of symbiont cellular deterioration. The potential sites of host mitochondrial disruption were also assessed by measuring changes in the expression of genes associated with electron transport and ATP synthesis using quantitative RT-PCR. The primary site of degradation appeared to be downstream of complex III of the electron transport chain with a significant reduction in host cytochrome c and ATP synthase expression. The consequences of reduced expression could limit the capacity of the host to mitigate ROS generation and maintain both organelle integrity and cellular energy supplies. The disruption of host mitochondria, cellular homeostasis, and subsequent cell death irrespective of symbiont integrity highlights the importance of the host response to thermal stress and in symbiosis dysfunction that has substantial implications for understanding how coral reefs will survive in the face of climate change.     

Initial events of light-dependent ATP synthesis in spinach subchloroplast particles.

The kinetics of 32Pi incorporation into adenine nucleotides by subchloroplast particles in the light is studied with a continuous flow apparatus allowing measurements between 3 and 200 ms. After a short lag time from 1 to 3 ms ATP synthesis proceeds with a constant rate. During the first few milliseconds a faster labelling of ADP is detected. This labelling of ADP reaches a constant level up to 1 molecule ADP labelled per molecule of coupling factor present. The labelling pattern in ATP indicates that the labelled ADP does not equilibrate with free ADP. The addition of 32Pi to a phosphorylating system during the light phase (32Pi pulse) exhibits unchanged kinetic characteristics for labelling of ATP and ADP. These results indicate a phosphorylation of AMP to ADP being an intermediate step in photophosphorylation. In experiments carried out in the dark no label is found in ATP within the time analysed. However the labelling of ADP occurs in the same way as in the light.     

Control of chlorophyll synthesis in Chromatium vinosum

Summary The role of adenine nucleotides in the control of the energy metabolism of Chromatium has been studied through the measurement of the levels of ATP ADP, and AMP in growing cultures. Comparison of these levels with the cellular chlorophyll levels indicates that ATP concentration, and not those of ADP, AMP, or any function of concentration of these three nucleotides, is a controlling factor in chlorophyll synthesis. In addition, the relationship of cellular ATP and chlorophyll levels to sulfur metabolism furnishes further evidence for the existence of two photosystems, postulated previously. The effects of high levels of exogenous ATP and ADP are consistent with these findings.     

Visualization of spatiotemporal energy dynamics of hippocampal neurons by mass spectrometry during a kainate-induced seizure

We report the use of matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry combined with capillary electrophoresis (CE) mass spectrometry to visualize energy metabolism in the mouse hippocampus by imaging energy-related metabolites. We show the distribution patterns of ATP, ADP, and AMP in the hippocampus as well as changes in their amounts and distribution patterns in a murine model of limbic, kainate-induced seizure. As an acute response to kainate administration, we found massive and moderate reductions in ATP and ADP levels, respectively, but no significant changes in AMP levels--especially in cells of the CA3 layer. The results suggest the existence of CA3 neuron-selective energy metabolism at the anhydride bonds of ATP and ADP in the hippocampal neurons during seizure. In addition, metabolome analysis of energy synthesis pathways indicates accelerated glycolysis and possibly TCA cycle activity during seizure, presumably due to the depletion of ATP. Consistent with this result, the observed energy depletion significantly recovered up to 180 min after kainate administration. However, the recovery rate was remarkably low in part of the data-pixel population in the CA3 cell layer region, which likely reflects acute and CA3-selective neural death. Taken together, the present approach successfully revealed the spatiotemporal energy metabolism of the mouse hippocampus at a cellular resolution--both quantitatively and qualitatively. We aim to further elucidate various metabolic processes in the neural system.     

Content of Adenosine Phosphate Compounds in a Long-Day Duckweed, Lemna gibba G3, under Different Light and Nutritional Conditions

The levels of ATP ADP and AMP as well as the energy charge were examined in the long-day duckweed. Lemna gibba G3, under different light and nutritional conditions. ATP and ADP content, but not AMP, decreased slightly when the plant was cultured with a medium depleted of sucrose or was exposed to continuous darkness. The energy charge was not affected either by changes in the light conditions or by the depletion of sucrose from the medium, although increase of fresh weight changed drastically under these conditions. The levels of ADP and AMP and the value of the energy charge remained nearly constant throughout a 1-day period when the plant was exposed to short-day, continuous light and continuous dark. ATP content decreased gradually during the light period of the short day and thereafter remained constant during the dark period. When the plant was exposed to continuous light. ATP content decreased until the 8th hour after beginning of the continuous light, only to recover rapidly to its original level during the phase coinciding with the preceding dark period of the short day. Under continuous dark conditions the ATP content remained constant throughout the day. It was concluded that the diurnal rhythm of physiological activities previously reported cannot be related to the energy charge.     

Short term cadmium intoxication of the shrimp Palaemon serratus: Effect on adenylate metabolism

• 1.1. Shrimps were exposed for 96 hr to various concentrations of cadmium under laboratory conditions. The LC₅₀ was around 4 ppm Cd in water, which corresponded to 0.180 μg/g wet weight of cadmium in tail muscles.
• 2.2. The effect of various concentrations of cadmium on adenylates was analyzed in tail muscles: At subletal cadmium concentrations, no variation of ATP, ADP and of the adenylate sum occurred, while the AMP concentration began to decrease from 0.06 ppm.

At the LC₅₀, the ATP, ADP and AMP concentrations dropped acutely, the ATP/ADP ratio increased acutely.The apparent equilibrium constant of the adenylate kinase reaction increased significantly from 2 ppm Cd, indicating an impairment in energetic metabolism.Cadmium intoxication did not influence the value of the adenylate energy charge.     

AMP-activated protein kinase: a cellular energy sensor with a key role in metabolic disorders and in cancer

It is essential to life that a balance is maintained between processes that produce ATP and those that consume it. An obvious way to do this would be to have systems that monitor the levels of ATP and ADP, although because of the adenylate kinase reaction (2ADP?ATP+AMP), AMP is actually a more sensitive indicator of energy stress than ADP. Following the discoveries that glycogen phosphorylase and phosphofructokinase were regulated by AMP and ATP, Daniel Atkinson proposed that all enzymes at branch points between biosynthesis and degradation would be regulated by adenine nucleotides. This turned out to be correct, but what Atkinson did not anticipate was that sensing of nucleotides would, in most cases, be performed not by the metabolic enzymes themselves, but by a signalling protein, AMPK (AMP-activated protein kinase). AMPK occurs in essentially all eukaryotes and consists of heterotrimeric complexes comprising catalytic α subunits and regulatory β and γ subunits, of which the latter carries the nucleotide-binding sites. Once activated by a metabolic stress, it phosphorylates numerous targets that alter enzyme activity and gene expression to initiate corrective responses. In lower eukaryotes, it is critically involved in the responses to starvation for a carbon source. Because of its ability to switch cellular metabolism from anabolic to catabolic mode, AMPK has become a key drug target to combat metabolic disorders associated with overnutrition such as Type?2 diabetes, and some existing anti-diabetic drugs (e.g. metformin) and many 'nutraceuticals' work by activating AMPK, usually via inhibition of mitochondrial ATP production. AMPK activators also potentially have anticancer effects, and there is already evidence that metformin provides protection against the initiation of cancer. Whether AMPK activators can be used to treat existing cancer is less clear, because many tumour cells appear to have been selected for mutations that inactivate the AMPK system. However, if we can identify the various mechanisms by which this occurs, we may be able to find ways of overcoming it.     

A kinetic study of the rat liver adenosine kinase reverse reaction

Adenosine kinase is an enzyme catalyzing the reaction: adenosine + ATP --> AMP + ADP. We studied some biochemical properties not hitherto investigated and demonstrated that the reaction can be easily reversed when coupled with adenosine deaminase, which transforms adenosine into inosine and ammonia. The overall reaction is: AMP + ADP --> ATP + inosine + NH(3). The exoergonic ADA reaction shifts the equilibrium and fills the energy gap necessary for synthesis of ATP. This reaction could be used by cells under particular conditions of energy deficiency and, together with myokinase activity, may help to restore physiological ATP levels.     

A Kinetic Study of the Rat Liver Adenosine Kinase Reverse Reaction

Adenosine kinase is an enzyme catalyzing the reaction: adenosine + ATP → AMP + ADP. We studied some biochemical properties not hitherto investigated and demonstrated that the reaction can be easily reversed when coupled with adenosine deaminase, which transforms adenosine into inosine and ammonia. The overall reaction is: AMP + ADP → ATP + inosine + NH₃. The exoergonic ADA reaction shifts the equilibrium and fills the energy gap necessary for synthesis of ATP. This reaction could be used by cells under particular conditions of energy deficiency and, together with myokinase activity, may help to restore physiological ATP levels.     

Insect-microbe mutualism without vertical transmission: a stinkbug acquires a beneficial gut symbiont from the environment every generation

The broad-headed bug Riptortus clavatus (Heteroptera: Alydidae) possesses a number of crypts at a posterior midgut region, which house a dense population of a bacterial symbiont belonging to the genus Burkholderia. Although the symbiont is highly prevalent (95 to 100%) in the host populations, the symbiont phylogeny did not reflect the host systematics at all. In order to understand the mechanisms underlying the promiscuous host-symbiont relationship despite the specific and prevalent association, we investigated the transmission mode and the fitness effects of the Burkholderia symbiont in R. clavatus. Inspection of eggs and a series of rearing experiments revealed that the symbiont is not vertically transmitted but is environmentally acquired by nymphal insects. The Burkholderia symbiont was present in the soil of the insect habitat, and a culture strain of the symbiont was successfully isolated from the insect midgut. Rearing experiments by using sterilized soybean bottles demonstrated that the cultured symbiont is able to establish a normal and efficient infection in the host insect, and the symbiont infection significantly improves the host fitness. These results indicated that R. clavatus postnatally acquires symbiont of a beneficial nature from the environment every generation, uncovering a previously unknown pathway through which a highly specific insect-microbe association is maintained. We suggest that the stinkbug-Burkholderia relationship may be regarded as an insect analogue of the well-known symbioses between plants and soil-associated microbes, such as legume-Rhizobium and alder-Frankia relationships, and we discuss the evolutionary relevance of the mutualistic but promiscuous insect-microbe association.